Neuroblastoma is one of the most common extra cranial solid tumors of pediatric population. It arises from neural crest cells and is commonly present in the adrenal medulla or along the sympathetic chain. Neuroblastoma cells have spontaneous morphologic differentiation. They can be differentiated into three different cell types- 1)Neuroblastic or N- type, 2)Schwannian/glial or S type, 3)Intermediate or I-type. S-cells are substrate adherent and non tumorigenic in soft agar, whereas I cells are highly invasive and malignant. I-type cells are the multipotent embryonic precursors capable of giving rise to both N and S cells. Invasive tumor cells have the ability of migration and angiogenesis, besides abnormal growth and proliferation. Epidermal Growth Factor Receptors (EGFRs) play an important role in cell survival, growth and proliferation of normal and malignant cells and also stimulate angiogenesis, invasion/metastasis and inhibition of apoptosis in tumor cells. Autotaxin, an ectonucleotide pyrophosphatase phosphodiesterase (ENPP2) is a mitogen responsible for cell motility and angiogenesis in some types of tumor cells. Autotaxin also confers tumorigenic and metastatic potential to some types of cancers.
Figure 1-Primers of EGFR (NM_ 005228.3) and ENPP2 (NM_006209.3) genes used in this project and characteristics of the PCR products generated.
Figure 2-Expression of Epidermal Growth Factor Receptor (EGFR) in 2 different neuroblastoma cell lines- SH- EP1(S-type) and SK-N-ER (I-type). EGFR primers were located in exons 27and 28 and designed to amplify a segment of 265 bp. GAPDH was run as a control and produced an amplified segment of 228 bp. RT-PCR products were fractionated on 1.5% agarose gel with ethidium bromide and visualized under UV light.
Figure 3-Expression of ENPP2 in SH-EP1 (S-type) and SK-N-ER (I-type) neuorblastoma cell lines. ENPP2 primers were located in exons 12 and 16 and designed to amplify a segment of 462 bp. GAPDH was run as a control and produced a 228 bp product. RT-PCR products were subjected to 1.5% agarose gel with ethidium bromide and visualized under UV light
Figure 4-(A) Partial sequence alignment of 265 bp product with reported EGFR mRNA (NCBI Accession Number NM_005228.3). About 160 nucleotides of the purified RT-PCR product shows sequence homology with the reported EGFR mRNA & (B) Partial sequence alignment of 462 bp product with reported ENPP2 mRNA (NCBI Accession Number NM_ 006209.3). About 160 nucleotides of the purified RT-PCR product shows sequence homology with the reported ENPP2 mRNA
The expression levels of EGFR and Autotaxin (ENPP2) in S and I- type neuroblastoma cell lines were studied by RT-PCR analysis using specific primers for human EGFR and ENPP2 mRNA.
The results of RT-PCR showed that EGFR encoding RNA has been equally expressed in both S and I- type neuroblastoma cell lines. On the other hand, Autotaxin (ENPP2) encoding RNA was found to be expressed only in I but not in the S-type cell line.
Sequence alignment of 265 bp RT-PCR product with reported human EGFR mRNA and 462 bp RT-PCR product with reported human Autotaxin (ENPP2) mRNA showed high sequence homology.
From the present study, it appears EGFR expression is not related to malignancy in neuroblastoma. However, analysis of larger sample size is required to reach any definitive conclusion.
Future research is needed to evaluate the expression level of Autotaxin (ENPP2) mRNA and protein in larger number of neuroblastoma cell lines to determine the significance of the observation made by this study.
Please refer to the attached full paper for more details
I would like to thank Dr. Berish Y. Rubin for his guidance and for giving me the opportunity to pursue this project. Special thanks to Joseph Frezzo and Leelesha Samaraweera for their constant patience, tireless support and help. I would also like to acknowledge Dr. Robert R. Ross and Barbara Spengler for kindly providing neuroblastoma cell lines.
|This document was last modified 05/09/2007.|
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